by a team consisting of the Core Leader (Dr. W.C. Stanley), the Core Co-Leader (Dr. C.L. Hoppel), two Co-Investigators (Drs. J. Kemer and M.P. Chandler) and numerous Research Assistants (Ms. H. Huang and T.A. McElfresh, Dr. W. Parland and Mr. J. Sterk, and E. Vasquez). The work flow in this Core is directly linked to the work in Projects 1 through 4, as set forth in the Specific Aims of each Project. The Specific Aims are distributed over the five years of the grant as depicted in the scheme on the following page. JJ Principal InvestigatorfProgram Director(Last, first, middle): Stanley, William C. The distribution of blood and tissue samples, and illustrated in Figure 1. The Metabolism core will microembolization-induced HF dogs used in Projects dogs used in Project 3. the analyses performed by the metabolic core are analyze blood and tissue samples from coronary 1, 2 and 4, and from the rapid pacing-induced HF Figure 1. Uses of the Metabolic Core Conduct terminal metabolic studies P-RpOroJvEidCeT bl1ood and i tissue samples for analysis PROJECT 2 Provide HF dogs for terminal metabolic studies-all samples will be analyzed by the core PROJECT 3 Provide blood and tissue samples for oxidation and mitochondrial assessments PROJECT 4 Provide myocardium from by Individual Projects: METABOLISM CORE -in vivo substrate oxidation assessment -mitochondrial isolation and basic ETC analysis. -enzyme activities and expression -CoAs and carnitines -tissue preparation for histology PROJECT 4 Obtain mitochondria for detailed analysis of respiratory function and complexes HI and IV Project 1: All specific aims of Project 1 will require metabolite and isotopic tracer analysis in blood 13 3 a 14 and tissue (eg C-lactate, H-oleate, and C-glucose in blood); assessment of the expression and function of selected proteins (PDH, CPT-I and II, MCAD, etc); isolation of mitochondria and assessment of mitochondrial function; and measurement of the content of key regulatory metabolites (e.g. long and short chain CoA esters and camitines, ATP, and lactate). Project 2: All animals randomized to Specific Aims 1-3 will be shipped to Dr. Stanley's laboratory in Cleveland for terminal metabolic studies as described in Specific Aim 1 of Project 1. These studies will measure, LV function, myocardial glucose, lactate and free fatty acid oxidation, the activity and/or expression of key enzymes, the content of key regulatory metabolites, the isolation ofmitochondria and assessment ofmitochondrial function, all under basal conditions. All these analyses will be performed by the Metabolic Core. Project 3: Left ventricular tissue samples harvested from HF involved in Specific Aims 1-3 will be shipped Core. Analyses will include substrate concentrations FFA oxidation (CPT-I and MCAD) and glycolytic Cycle enzyme, citrate synthase. Tissue from dogs isolation of both populations of mitochondria and function. normal dogs and dogs with pacing induced to Cleveland for analysis in the Metabolic and oxidation analyses, enzymes of the (GAPDH and PDH) pathway and Krebs with pacing induced HF will require assessment of mitochondrial respiratory JJ Principal Investigator/Program Director (Last, first, middle): Stoxlley, William C. Project 4: All animals that undergo metabolic studies (Project 1 and 2, Aims 1-3) will have fresh LV tissue processed in the core for mitochondrial isolation and mitochondrial respiration studies. These mitochondria will be analyzed by Project 4 for oxidative phosphorylation, and detailed analysis of complex III and IV function. In addition, Project 4 will provide the Core with myocardium from dogs with pacing-induced HF for mitochondrial isolation and ETC activity measurements, and subsequent analysis of isolated mitochondria by Project 4 for oxidative phosphorylation, and detailed analysis of complex III and IV function. B. BACKGROUND AND SIGNIFICANCE Over the past 5 years, Drs. Stanley and Hoppel have fully developed and validated all of the biochemical methods for use on canine myocardium as they are outlined in this Metabolism Core. Dr. Stanley has 17 years of experience with in vivo measurement of substrate metabolism using isotopic tracer in humans, pigs and dogs. Dr. Hoppel has been continuously performing mitochondrial isolation and function studies for over 35 years, and his laboratory routinely performs function analysis of mitochondria (oxidative phosphorylation, ETC complex activities, citric acid cycle function, fatty acid oxidation enzymes, etc.) for the Center for Inherited Diseases of Energy Metabolism. Dr. Stanley's laboratories will perform the metabolic and isotopic tracer analysis of blood and tissue samples from dogs with rnieroembolization-induced HF from Projects 1, 2, 3 and 4. These techniques have been used extensively by Dr. Stanley and many papers published from his laboratory will document the use of these methods as outlined in this Metabolism Core (1-9). Dr. Hoppel's laboratory will isolate mitochondria and assess mitochondrial function, determine the activity of the electron transport chain complexes in isolated heart mitochondria, assess the expression and function of selected proteins (PDH, CPT-I and II, MCAD, etc); and measure the content of key regulatory metabolites (e.g long and short chain CoA esters and carnitines, ATP, and lactate). Numerous publications published from his laboratory document the use of these methods (10-17). Dr. Hoppel's experience has been mostly with rat heart mitochondria, although they have prepared mitochondria from mouse, hamster, beef and dog. Recent data for dogs with HF are given in the preliminary results section below. C. PRELIMINARY RESULTS C1. Activity and regulatory properties of CPT-I Using the canine model of coronary microembolism-induced HF (18) we have shown that despite the decreased left ventricular ejection fraction there was no change in maximal activity of CPT- I, the rate controlling enzyme of overall mitochondrial fatty acid oxidation, medium-chain acyl-CoA dehydrogenase and citrate synthase when compared to control dogs (9). Since both the MCAD and citrate synthase activities are unaltered between the two groups, there is no obvious change in the function of either 13-oxidation or the citric acid cycle. These data are superficially in conflict with studies by Paolissio et al. (19) suggesting increased fatty acid oxidation in HF and with those reporting a decreased fatty acid oxidation (8). However, a possible explanation of these apparently discrepant findings could lie, in addition to the different species used, either in changes in tissue malonyl-CoA concentrations or changes in the sensitivity of CPT-I to malonyl-CoA inhibition. Indeed, in humans with congestive HF, while there is no change in CPT-I activity, there is a significant increase in IC50 value of malonyl-CoA. Although no effort has been made to elucidate the mechanism for changes in malonyl-CoA sensitivity, the authors speculate that a shift from the muscle isoform to the liver isoform JJ Principal Investigator/Program Director (Last, first, middle): Stanley, William C. could be the reason for this (20). Alternatively, differences in experimental findings also might arise if animals in different stages of HF were used. As a follow-up on the studies on canine heart homogenates and to test whether changes in the regulatory properties of CPT-I could contribute to the proposed switch in fuel selection in HF, we used isolated mitochondria to determine the maximal CPT-I activity as well as the enzyme's sensitivity to malonyl-CoA inhibition. Since the two mitochondrial populations present in heart could be affected to a different extent, as documented for the cardiomyopathic Syrian hamster (16), both, subsarcolemmal (SSM) and interfibrillar mitochondria (IFM) have been studied. The two populations ofmitochondria were isolated from hearts of control and HF dogs using the procedure as outlined in the Analytical Methods. The mitochondrial protein yield for two mitochondrial populations are presented in Table 1. Table 1: Mitochondrial protein yield for subsarcolemmal (SSM) and interfibrillar mitochondria (IFM) isolated from control and HF dogs. Control I:IF SSM 5.32 + 0.71" 4.54 + 0.66* IFM 10.99 + 0.45 8.36 + 0.80 _ The values represent the mean + SEM of nine separate animals. The protein yield is significantly different * = P < 0.05 between SSM and IFM in both groups of animals. & = P < 0.05 between IFM of control and I-IF _nimals. The protein yield of SSM was significantly lower than that oflFM in both control and HF dogs. While the protein yield of IFM was significantly decreased in HF (24% decrease vs. control), the difference in SSM yield (15% decrease vs. control) was not significant. The decreased mitochondrial yield suggests that there is a decrease in mitoehondrial density even at this relative mild stage of HF. As shown in Figure 2, IFM have significantly higher CPT-I activity as compared to SSM. However, the maximal activity of this enzyme is not affected in either mitochondrial population by HF. "_ 40_ 1- [unreadable],-: 32 1 _ 24 t C _ 16 E C _I:-III _ 8 ,, _ 0_ 0 0 - SSM IFM Fi2ure 2. CPT-I specific activity of cardiac subsasrcolemmal (SSM) and interfibrillar mitochondria (IFM) isolated from control and microembolism induced heart failure dogs. The values represent the mean + SEM of nine separate mitochondrial preparations in each group. 267 JJ Principal Investigator/Program Director (Last, first, middle): Stanley, William C. Asterisks denote a signicant (P < 0.05) difference in CPT-I activity between SSM and IFM in both groups of animals. To evaluate whether HF induced changes in the enzyme's regulatory properties, we determined the IC50 values ofmalonyl-CoA for CPT-I on isolated SSM and IFM. The data presented in Figure 3 and Table 2 show that dog heart mitochondrial CPT-I is extremely sensitive to malonyl-CoA inhibition with an IC50 value of 0.07 - 0.1 lpM. Approx. 80-85% becomes inhibited by as low as 0.4/aM malonyl-CoA with the remaining CPT-I activity being unaffected at least in the concentration range reported for different species (up to 2.01,tM). Whether the CPT-I activity not inhibited by physiological concentrations ofmalonyl-CoA represent the less sensitive liver isoform or represents a posttranslationaly modified muscle isoform remains to be elucidated. _o25 Dog,heart SSM [unreadable]"o= 35 Dog h_lFM _20 -..!.- (3:xlt. [unreadable]-28 --I-0::_. [unreadable]1 E 15 _I-F E 21 O) or) 10 14 SD :D E 5 E 7 ! ! I-" I-- o.. 0 o.. 0 O O 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 [M_lonyi-CoA]uM [Malonyl-CoA]uM Figure 3. Determination of IC50 values ofmalonyl-CoA for CPT-I on cardiac subsarcolemmal (SSM) (left panel) and interfibrillar mitochondria (IFM) (fight panel) isolated from control (Cont.) and microembolism induced (HF) dogs. The values represent the mean + SEM of nine separate mitochondrial preparations in each group. Table 2:IC50 values ofmalonyl-CoA for CPT-I in subsarcolemmal (SSM) and interfibriUar mitochondria (IFM) isolated from heart of control and HF dogs. Control HIE SSM 0.072 + 0.011 0.107 _+0.024 IFM 0.077 + 0.011 0.088 + 0.011 The values represent the mean + SEM of nine separate animals. There are no differences in the IC50 values between SSM and IFM and these values are not affected by HF. Furthermore, since the inhibitor malonyl-CoA and the substrate palmitoyl-CoA are mutually competitive, the lack of changes in IC50 also suggests that the Km values for palmitoyl-CoA are not affected by HF either. In line with these in vitro data on dog heart homogenates and isolated dog heart mitochondria, no changes in either long-chain fatty acid oxidation or glucose oxidation were observed in perfused hearts of HF dogs (see preliminary data presented in Project 1). C2. Mitochondrial substrate utilization JJ Principal Investigator/Program Director (Last, first, middle): Stanley, William C. To determine whether the capacity of mitochondria to oxidize different substrates is affected by HF, we measured the oxygen consumption of isolated SSM and IFM. Glutamate, an electron donor via glutamate dehydrogenase and NADH, was used as a substrate to test the ETC (Figure 4). Pyruvate and malate was used to test the tricarboxylic acid cycle in addition to the ETC (Figure 4) and palmitoyl- CoA plus camitine and palmitoylcarnitine (Figure 5) was used to assess the CPT-I-dependent and CPT-I independent aspect ofmitochondrial [3-oxidation, respectively. As shown in Figures 4 and 5, in SSM there was a significant decrease in 2mM ADP-stimulated respiration with all four substrates tested. While a decrease in respiration was also observed in IFM, this did not reach statistical significance (P= 0.06-0.09). No differences in state 4 respiratory rates and ADP/O ratios were observed (data not shown). Pyruvate Glutamate T rl HF SSM IFM Figure 4. Maximal ADP-stimulated respiration mitochondria (IFM) isolated from control and glutamate/male (fight panel) as substrates. The seven (HF) separate experiments. Asterisks(*) denote a P value < 0.05. PalmitoyI-CoA [] Cont. _250. , T QFF E200. E 1 o. E 100- < e.- 0 SSM IFM 13HF E 9240 E o SSM IFM of cardiac subsarcolemmal (SSM) and interfibrillar HF dogs with pyruvate/malate (left panel) and data represent the mean + SEM of eight (control) and Palmitoylcamitine T n Cont. "_ E 150 IFM Fieure 5. Maximal ADP-stimulated respiration of mitochondria (IFM) isolated from control and HF panel) and palmitoylcamitine/malate (right panel) eight (control) and seven (HF) separate experiments. cardiac subsarcolemmal (SSM) and interfibrillar dogs with palmitoyl-CoA/camitine/malate (left as substrates. The data represent the mean _+SEM of JJ Principal Investigator/Program Director(Last, first, middle): Stanley, William C. Asterisks(*) denote a P value < 0.05. The respiratory data presented above strongly suggest that in HF there is an impaired mitochondrial metabolism due to decreased capacity of the respiratory chain. This conclusion is based on the fact that the oxygen consumption with the complex I (and with fatty acids, also flavin- dependent coenzyme Q reduction) substrates tested are affected to the same extent in SSM. C.3 Anall_sis of Ac_lcarnitines. Isolation of acylcamitines from rat heart was performed using silica gel solid-phase extraction columns. Acylcarnitines were then derivatized with pentafluorophenacyl trifluoromethane sulfonate and analyzed by HPLC/MS. Figure 6 shows the acylcarnitine profile of a rat heart perfused in the working mode with palmitate deuterated in the c0-methyl position (M+3). In addition to palmitoylcarnitine, significant amount of stearoylcamitine was found. Furthermore, smaller amounts of hydroxy-acylcamitines (incompletely oxidized fatty acid oxidation intermediates) were detected. O 5' O: 0 5 10 15 20 25 30 35 40 45 50 55 60 Time (min) Figure 6: Separation (HPLC) and identification (ESI-MS) of acylcamitines of a rat heart perfused with deuterated [C'H3] palmitic acid in the working mode. JJ c-E- 6-o_.3 d3C16 611.3 C18 636.4 I Species rn/z i d3C18 639.4 5' 0 52.0 52,5 53.0 53.5 Figure 7 Deuterium enrichment deuterated [C2H3] palmitic acid. Species m/z C12 552.3 d3C12 555.3 C14 580.3 d3C14 583.3 HO-C14 596.3 HO-d3C14 599.3 !,o-:t HO-C16 624.3 HO-d3C16 627.3 J*=l Principal Investigator/Program Director (Last, first, middle): Stanley, William C. 8 611,3 i_20i-t l _o_,,.,,.,,.,_, ,. i | iiii1| _1| i 590 600 610 _ 630 nYz 54.0 54.5 55.0 55.5 56.0 56,5 57.0 57.5 58.0 58.5 59.0 59.5 Time(rain) of palmitoyl- and stearoylcarnitine following perfusion of rat heart with [unreadable]! 4O 610 620 630 640 650 m/z j2 t ., t. .. h ,,,,l,_,,_',,,,l',,,_ [unreadable]] 560 570 580 590 60( *l=_ =,ill 580 590 600 610 620 rr_ lo9o:_: \ _z 1oo 5_ / \ !.o] / - ._ so: \ i o_,'_,,,.'.;,Jll.,.,'.';',I,", "_ _ 53o s4o sso ._o ST,: I 40 nVz 43 44 Figure 8: Deuterium perfusion of rat heart Data in Figures acylcamitines. 45 46 47 48 49 50 51 52 Tkne (rain) enrichment of fatty acid oxidation intermediates (3-hydoxyacylcamitines) following with deuterated [C2Ha] palmitic acid. 6-8 document the capability of this method to separate, identify and quantitate tissue 271 JJ Principal Investigator/Program Director (Last, first, middle): Stanley, William C. C.4 Malonvl Coil Analysis We will study the myocardial malonyl-CoA content in relation to CPT-I activity. Existing HPLC/UV methods for malonyl-CoA detection were modified, adding detection by ESIfMS (Figure 9 ). 10 Full Scale = 0.140 AU MS !: _8c 8O Chromatogramroo._ooomz _=7c UV =>5o Chmmatogram25_ m 3t [unreadable]3(: 21 oi ............ 1( G 0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10 Time (rain) Time (mJn) 259.0 lO[unreadable] 76 t "2 j8oi _1 MS Spectrum UV Spectrum_9o.4oo_ 808.3 4o! M MS Spectrumss,_z [unreadable]50 20_ O' .' ;..7. .... i"''-l'-'T-'',-" ""'T""'"'"'""" '-"T-'-F-'F''"'-"-'"| "-" ,----r--,-- -, I 200 250 300 350 400 wavelength (nm) Figure 9: HPLCRJV As shown in of malonyl-CoA. For and detection will be in figure 10. 1 (left panel) and HPLC/MS (fight panel) chromatogram of malonyl-CoA. Figure 9 dual detection (UV and MS) may allow for anticipated qualitative characterization quantification, stable isotope labelled malonyl-CoA will be used as the internal standard by selected reaction monitoring (SRM) MS. MS chromatograms and spectra are displayed A MS/MS Chromatogram ncss2.2 _ MS/MS Chromatogram_ 2 0.0 0,5 1,0 1,5 2.0 2,5 3.0 3.5 4.0 4.5 5.0 5,5 6.0 6.5 7.0 Time (rain) 54 .1 _4[unreadable]1 ao_ o ............... 100-1 8 0.1 _s[unreadable]l / t / 300 400 500 600 700 800 nYz Figure 10: HPLC chromatogram of unlabelled and 13C3-malonyl-CoA 272 7,5 8.0 8,5 g.O 9,5 10,0 MS/MS Spectrume_._r_z . MS/MS Spectrumess'_nn 900 1000 (M+3) with ESI/MS detection. JJ The major advantage of this method malonyl-CoA by SRM and the stable isotope RT:a.LI0 - e.O f.}0 50 _CoA I_ Jl I,_1-1 _ll I, I I J J J I'lll'J I'; IJ I I' II'lll I'l 4 5 6 7 8 9 10 11 Principal Investigator/Program Director (Last, first, middle): Stanley, William C. over existing HPLC methods labelled internal standard for til I_ '1J m I Jl Ill III I I _1 _ I 12 13 14 15 16 17 18 is the highly selective detection of quantification. UV254nm MSTIc7OO-lOOOm/z MSRIc852r_ MSRIc766m/z ' 19 20 Time (rain) Figure 11. HPLC separation with UV and ESI/MS detection of rat liver extract. The top chromatogram is the UV chromatogram, showing several UV absorbing peaks. The second chromatogram is the total ion current chromatogram, which shows MS response for some but not all of the UV absorbing peaks. The remaining chromatograms are reconstructed ion chromatograms 0tIC), which identify UV and MS chromatographic peaks corresponding to malonyl- CoA, CoA and acetyl-CoA, respectively. Of great interest is the UV chromatographic peak corresponding to malonyl-CoA. The UV response is much greater than the MS response, indicating that there is UV absorbing material co-chromatographing with malonyl-CoA which is not malonyl- CoA. The sensitivity of the analysis has been increased by employing on-line HPLC trapping of malonyl-CoA followed by microbore HPLC chromatography and is sufficiently sensitive to accurately quantitate malonyl-CoA in rat heart and liver. C.5 Phospholipid and Cardiolipin Analwis: Methods for separation, quantitation and characerization of mitochondrial phospholipids had been worked out in Dr. Hoppel's laboratory (21-23) and are used regularly. We have obtained data documenting that cardiolipin is modified following ischemia in the aged heart mitochondria. Using methods referenced above, cardiolipin was isolated from rat heart mitochondria by a silica gel column chromatographic class separation of lipids followed by normal phase high